Mitsuharu Hasegawa

Inductive Effects of Dexamethasone on the Gene Expression of Cbfa1, Osterix and Bone Matrix Proteins During Differentiation of Cultured Primary Rat Osteoblasts

Runx2/core binding factor alpha 1 (Cbfa1) and Osterix (Osx) are osteoblast-specific transcription factors essential for the development of a mature osteoblast phenotype and are thought to activate osteoblast marker genes in vivo to produce a bone-specific matrix. Dexamethasone (Dex) is known to be a potent stimulator of osteoblastic differentiation in vitro, however, the exact role is still unclear. To investigate the mechanisms of the stimulation of osteoblastic differentiation by Dex, we evaluated the effects of Dex on proliferation and mineralization as well as on mRNA expression of Cbfa1, Osx and osteoblast marker genes, osteocalcin (OC) and bone sialoprotein (BSP) mRNAs in differentiating foetal rat calvarial cells (FRCC), which were cultured for 35 days in the presence or absence of 10−7 M Dex. Treatment of FRCC with Dex resulted in the stimulation of cell proliferation and increased the number of cells, which are able to produce bone-like nodules with a mineralized matrix when compared to untreated controls. Northern blot analysis revealed that, in the absence of Dex, Cbfa1 mRNA expressed at day 8, while Osx mRNA expressed at day 15. Subsequently expression of these mRNAs increased up to day 21, followed by constant expression during the culture period. The expression of OC and BSP mRNAs appeared to be synchronous with that of Osx mRNA and was detectable at day 15 with an increase thereafter. The presence of Dex resulted in an induction in Cbfa1 and Osx mRNA expression. The former appeared at day 5 and the latter appeared at day 11. Subsequently expression of Cbfa1 and Osx mRNAs increased up to day 15 with a decrease thereafter. Expression of OC and BSP mRNAs appeared to be coincident with that of Osx mRNA and was detectable at day 11 and reached a maximum at day 15 followed by constant expression. These observations indicate that induction of Cbfa1 and Osx mRNAs by Dex may be followed by activation of osteoblast marker genes such as OC and BSP mRNAs to produce a bone-specific matrix that subsequently becomes mineralized. Thus, it is likely that Dex may promote osteoblastic differentiation and mineralization of FRCC by inducing the expression of Cbfa1 and Osx genes in vitro.

Effects of bone morphogenetic protein-2 and transforming growth factor beta1 on gene expression of transcription factors, AJ18 and Runx2 in cultured osteoblastic cells.

Osteoblast differentiation is controlled by multiple transcription factors, Runx2, AJ18, Osterix, Dlx5 and Msx2. The mechanisms of regulation of AJ18 mRNA expression by the transforming growth factor β (TGF-β) superfamily remain poorly understood. However, it is known that BMP-2 induces differentiation of C26 cells into more mature osteoblastic cells. The present study, using Northern blot and real-time reverse transcription polymerase chain reaction analyses, investigated the effects of bone morphogenetic protein-2 (BMP-2) and TGF-β 1 on mRNA expression of AJ18 and Runx2 in a clonal osteoblast precursor cell line ROB-C26 (C26) cultured for 3, 6 or 9 days in the presence or absence of BMP-2. Although mRNA expression of Osterix and bone sialoprotein (BSP) was undetectable in the C26 culture, BMP-2 induced Osterix expression on days 3–9, but not BSP expression. BMP-2 also stimulated significantly Dlx5 expression on days 3–9, Msx2 and matrix Gla protein expressions on days 3 and 6, Runx2, alkaline phosphatase and osteocalcin expressions on days 6 and 9 in the culture. Furthermore, BMP-2 increased significantly Smad5 mRNA in the culture on day 3, indicating BMP-2 involvement in the regulation of Smad5 mRNA expression. In contrast, the inhibitory effects of BMP-2 on AJ18 mRNA expression were significant on days 3–9, indicating that a decrease in AJ18 mRNA expression is essential for the increased osteoblastic differentiation. Furthermore, TGF-β 1 (0, 0.1, 1.0 and 5.0 ng/ml) treatment of C26 cells cultured for 6 days in the presence or absence of BMP-2 for 24 h stimulated mRNA levels of AJ18 and Runx2, maximal stimulation occurring principally at 1.0 ng/ml. These observations indicate that the expression of AJ18 and Runx2 mRNAs in C26 cells is under the control of BMP-2 and TGF-β 1, which exert different effects on AJ18 mRNA expression, but are potent stimulators of Runx2 mRNA expression during osteoblast differentiation.

Malignant lymphoma arising from postoperative benign lymphoepithelial lesion. Report of a case and review of literature.

患者は42歳女性。約3年前から両側耳下腺部が腫張し, その増大と口腔乾燥感の増強のため1993年7月当科を受診した。顔貌はび漫性に両側耳下腺部が腫脹し, 触診で, 左側では比較的境界明瞭な鶏卵大の弾力性やや硬の腫瘤1個, 右側では胡桃大の同様の腫瘤2個を触知した。各種臨床検査および画像所見でも腫瘤に対する診断を確定しえず, 耳下腺腫瘍の可能性も考慮して確定診断を得る目的で左側耳下腺浅葉切除術を施行した。2か月後右側耳下腺浅葉切除術を施行した。病理組織学的診断はいずれもBenign lymphoepithelial lesion (LEL) であった。術後約1年を経過し, 左側の耳下腺部に大豆大の腫瘤が出現し漸次増大傾向を示したため, 1995年2月生検を行いdiffuse medium sized cell lymphomaと診断された。その後駿河台日大病院内科へ転科し, CHOP療法を5クール, MEK-2療法4クールの化学療法を受け1997年まで経過観察中である。本症例は, Pan B cellマーカーであるCD20陽性で, TcellマーカーであるCD3は陰性を示したところから, 耳下腺のリンパ上皮性疾患を基盤として発生したMALT lymphomaと診断した。